1. Field of the Invention
The present invention relates to a dry immunoassay element in which a homogeneous enzyme immunoassay is utilized, and an immunoassaying process in which the dry immunoassay element is used.
2. Description of the Prior Art
Analyses of the constituents of the living body or plasma proteins or the like contained in the body fluids, such as, blood and urine, are useful for diagnosing the condition of diseases or judging the course of curing, and thus they occupy important parts in the field of clinical test. The so-called enzyme immunoassay has been known in the art as one method for analyzing such constituents (ligands) generally present in a small amount in the body fluids. The enzyme immunoassay may be classified into heterogeneous systems for which band/free (B/F) separation must be effected, and homogeneous system for which B/F separation is not necessary. The reactions in the homogeneous system are based on the phenomenon that the enzymatic activity of the labelling enzyme is affected by some interference caused by binding of an antibody to the antigen (ligand), and the inhibition due to antigen-antibody binding is generally utilized. It is believed that enzymatic activity is suppressed by a steric hindrance caused by binding the enzyme to the substrate or a change in three-dimensional structure of the enzyme, when the antibody which is generally a large molecule is bound to the antigen in the enzyme-labelled antigen.
A dry analysis method is known, in which a homogeneous enzyme immunological reaction is utilized (see Unexamined Japanese Patent Publication No. 80050/1986). This dry analysis method uses the following reagent composition:
(A) A water-insoluble high polymer substrate labelled with a dye; and
(B) A conjugate of an antibody against the ligand and an enzyme for the substrate.
A sample containing an antigen (ligand) is allowed to react with a predetermined amount of an enzymelabelled antibody for a predetermined time to form a complex of the antigen and enzyme-labelled antibody, and then a water-insoluble and dye-labelled high polymer substrate is added to the reaction mixture to initiate an enzymatic reaction.
The enzyme of the enzyme-labelled antibody which has not been bound with the antigen reacts with the water-insoluble high polymer substrate to produce a lower molecular weight dye-labelled product which is soluble in water. On the other hand, the complex of macromolecular antigen, antibody and enzyme cannot exhibit the enzymatic activity towards the high polymer substrate. Accordingly, as the quantity of the antigen in the sample is increased, the product produced by the enzymatic reaction decreases. The quantity of the lower molecular weight product is determined by measuring the optical density of the absorption produced by the colored moiety and the antigen in the sample is analyzed quantitatively.
However, in this method, since a high polymer substrate bound to a dye, such as a dye-starch, is used and the dye bound to amylose which is the decomposition product formed by the action of the enzyme (amylase) is determined, the high polymer substrate and the reaction product must be separated prior to the measurement or determination. This is problematic because complicated operations are required which prevent automation of the analysis system. On the other hand, in the routine clinical test wherein a number of test samples must be processed, the samples must be analyzed rapidly using a simple process preferably by automated operations. For this reason, dry analysis elements have been proposed (see, for example, Unexamined Japanese Patent Publication Nos. 53888/1974, corresponding to U.S. Pat. No. 3,992,158, 77356/1984, corresponding to EP 0097 952A and 102388/1974 and U.S. Pat. No. 4,459,358).
However, in this known analysis element, since a high polymer substrate bound to a dye, such as, a dye-starch, is used and the dye bound to amylose which is the decomposition product formed by the action of the enzyme (amylase) is determined, the high polymer substrate and the reaction product must be separated prior to the measurement or determination. It is, therefore, necessary to provide a light-shielding layer containing, for example, titanium dioxide fine particles between the reagent layer containing the unreacted substrate and the detection layer for receiving the reaction product. An analysis element having this laminated structure is not preferred because it to take too much time for the soluble reaction product formed in the reagent layer to be diffused through the light-shielding layer into the detection layer. As a result, a rapid quantitative determination, which is a major advantage of the dry analysis element, cannot be carried out.
It is possible to improve the diffusibility of the reaction product by introducing a hydrophilic group, such as, a carboxyl or sulfo group, into the substrate to accelerate the diffusion of the reaction product. However, sites for introduction of such substituting groups are limited. Also, the introduction of the group may lower the molecular extinction coefficient of the dye site governing the sensitivity of the analysis.
One object of this invention is to provide an immunoassay element utilizing a homogeneous enzyme immuno-assay for enabling the rapid analysis of an analyte at high sensitivity using a simple operation.
We have discovered that this can be achieved by use of an immunoassay element for quantitatively analyzing a macromolecular (high polymer) antigen by determining the change in enzymatic activity caused by a reaction between said macromolecular antigen and an enzyme-labelled anti-body, said element comprising a substrate layer containing a non-diffusible substrate which forms a diffusible material in the presence of said enzyme, and a reagent layer containing a decomposing enzyme for further decomposing said diffusible material into a lower molecular weight product. The enzyme-labelled antibody may be contained in the substrate layer or in another layer laminated on the substrate layer.
Another object of this invention is to provide a process for quantitatively analyzing an analyte while using the aforementioned immunoassay element.
This object may be achieved by determining the change in enzymatic activity caused by a reaction between the macromolecular antigen and an enzyme-labelled antibody, comprising the steps of:
(a) applying said sample on a substrate layer containing a non-diffusible substrate to form a diffusible material in the presence of said enzyme;
(b) allowing the diffusible material to migrate into a reagent layer containing a decomposing enzyme for decomposing the diffusible material into a lower molecular weight product; and
(c) measuring the amount of the lower molecular weight product formed in the reagent layer.
The enzymatic activity of the enzyme of the enzyme-labelled antibody, bound to the macromolecular antigen contained in the sample, towards the non-diffusible substrate is inhibited by steric hindrance. As a result, the quantity of the diffusible material formed in the substrate layer is inversely proportional to the quantity of the antigen contained in the sample. The diffusible material formed in the substrate layer migrates rapidly into the reagent layer, where it is further decomposed into a lower molecular weight product. The lower molecular weight product may be detected in a detection layer. The unreacted non-diffusible substrate is held in the substrate layer.